Concentrated aqueous silk fibroin solution and use thereof

ABSTRACT

The present invention provides for concentrated aqueous silk fibroin solutions and an all-aqueous mode for preparation of concentrated aqueous fibroin solutions that avoids the use of organic solvents, direct additives, or harsh chemicals. The invention further provides for the use of these solutions in production of materials, e.g., fibers, films, foams, meshes, scaffolds and hydrogels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/425,541 filed on Apr. 17, 2009, which is a continuation of U.S.patent application Ser. No. 11/247,358 filed on Oct. 11, 2005 and issuedas U.S. Pat. No. 7,635,755 on Dec. 22, 2009, which is a continuation ofInternational Application No. PCT/US2004/011199 filed on Apr. 12, 2004,which designated the U.S., and which claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Application No. 60/461,716 filed on Apr. 10,2003, and U.S. Provisional Application No. 60/551,186 filed on Mar. 8,2004, the contents of each of which are incorporated herein by referencein their entireties.

GOVERNMENT SUPPORT

This invention was supported by the NIH, the NSF and the Air Force(subcontract from Foster Miller) Grant Nos. R01EB003210,R01DE13405-01A1, DMR-0090384, F49620-01-C-0064. The government hascertain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 14, 2013, isnamed 700355-053849-C5_SequenceListing and is 1,367 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to methods for preparation ofconcentrated aqueous silk fibroin solutions and to the use of thesesolutions in the production of silk fibroin materials such as, fibers,films, sponge-like porous foams, 3-dimensional scaffolds, and hydrogels.In particular, an all-aqueous means for preparation of silk fibroinsolutions is described.

BACKGROUND OF THE INVENTION

Silk is a well described natural fiber produced by the silkworm, Bombyxmori, which has been used traditionally in the form of threads intextiles for thousands of years. This silk contains a fibrous proteintermed fibroin (both heavy and light chains) that form the thread core,and glue-like proteins termed sericin that surround the fibroin fibersto cement them together. The fibroin is a highly insoluble proteincontaining up to 90% of the amino acids glycine, alanine and serineleading to β-pleated sheet formation in the fibers (Asakura, et al.,Encylopedia of Agricultural Science, Arntzen, C. J., Ritter, E. M. Eds.;Academic Press: New York, N.Y., 1994; Vol. 4, pp 1-11).

The unique mechanical properties of reprocessed silk such as fibroin andits biocompatibility make the silk fibers especially attractive for usein biotechnological materials and medical applications. Silk provides animportant set of material options for biomaterials and tissueengineering because of the impressive mechanical properties,biocompatibility and biodegradability (Altman, G. H., et al.,Biomaterials 2003, 24, 401-416; Cappello, J., et al., J. Control.Release 1998, 53, 105-117; Foo, C. W. P., et al., Adv. Drug Deliver.Rev. 2002, 54, 1131-1143; Dinerman, A. A., et al., J. Control. Release2002, 82, 277-287; Megeed, Z., et al., Adv. Drug Deliver. Rev. 2002, 54,1075-1091; Petrini, P., et al., J. Mater. Sci-Mater. M 2001, 12,849-853; Altman, G. H., et al., Biomaterials 2002, 23, 4131-4141;Panilaitis, B., et al., Biomaterials 2003, 24, 3079-3085). For example,3-dimensional porous silk scaffolds have been described for use intissue engineering (Meinel et al., Ann Biomed Eng. 2004 January;32(1):112-22; Nazarov, R., et al., Biomacromolecules in press). Further,regenerated silk fibroin films have been explored as oxygen- anddrug-permeable membranes, supports for enzyme immobilization, andsubstrates for cell culture (Minoura, N., et al., Polymer 1990, 31,265-269; Chen, J., et al., Minoura, N., Tanioka, A. 1994, 35, 2853-2856;Tsukada, M., et al., Polym. Sci. Part B Polym. Physics 1994, 32,961-968). In addition, silk hydrogels have found numerous applicationsin tissue engineering, as well as in drug delivery (Megeed et al., PharmRes. 2002 July; 19(7):954-9; Dinerman et al., J Control Release. 2002Aug. 21; 82(2-3):277-87).

However, in order to prepare silk based materials described above,chemical agents or organic solvents, such as hexafluoroisopropanol(HFIP), have been used for cross-linking or for the processing (Li, M.,et al., J. Appl. Poly. Sci. 2001, 79, 2192-2199; Min, S., et al., Sen'iGakkaishi 1997, 54, 85-92; Nazarov, R., et al., Biomacromolecules inpress). For example, HFIP is used to optimize solubility of the silk andmethanol is used to induce an amorphous to β-sheet conformationtransition in the fibroin, in order to generate water-stable silkstructures.

The use of organic solvents in the preparation of silk fibroin materialsrepresents a significant drawback, as organic solvents posebiocompatibility problems when the processed materials are exposed tocells in vitro or in vivo. Organic solvents can also change theproperties of fibroin material. For example, the immersion of silkfibroin films in organic solvents such as methanol causes dehydration ofthe hydrated or swollen structure, leading to crystallization and thus,loss of solubility in water. Further, with respect to tissue engineeringscaffolds, the use of organic solvents can render the silk material tobe less degradable. Thus, there is a need in the art for the developmentof silk based materials that can be formed in the absence of chemicalcross-linking and/or organic solvents.

SUMMARY OF THE INVENTION

The present invention provides for concentrated aqueous silk fibroinsolutions and an all-aqueous mode for preparation of concentratedaqueous fibroin solutions that avoids the use of organic solvents orharsh chemicals. The invention further provides for the use of thesesolutions in production of materials, e.g., fibers, films, foams,meshes, scaffolds and hydrogels.

In one embodiment, an aqueous silk fibroin solution is provided that hasa fibroin concentration of at least 10 wt % and wherein said solution isfree of organic solvents. Also provided for are aqueous silk fibroinsolutions wherein the fibroin concentration is at least 15 wt %, atleast 20 wt %, at least 25 wt %, or at least 30 wt %. If desired, thesolution can be combined with a biocompatible polymer before processing.

The fibroin of the aqueous silk fibroin solution can be obtained from asolution containing a dissolved silkworm silk, e.g. from Bombyx mori, adissolved spider silk, e.g. from Nephila clavipes, or from a solutioncontaining a genetically engineered silk.

In one embodiment of the invention, the aqueous silk fibroin solutionsdescribed herein, further comprise a therapeutic agent. Therapeuticagents include, for example, proteins, peptides, nucleic acids and smallmolecule drugs.

In another embodiment, a method for the production of a concentratedaqueous fibroin solution is provided. The method comprises preparing anaqueous silk fibroin solution and dialyzing the solution against ahygroscopic polymer for a sufficient time to result in an aqueousfibroin solution of at least 10 wt %.

Hygroscopic polymers useful in the method of the present invention,include, for example, polyethylene glycol, amylase, or sericin.Preferably, the hygroscopic polymer is a polyethylene glycol (PEG) witha molecular weight of 8,000 to 10,000 g/mol. Most preferably, the PEGhas a concentration of 25-50%.

In one embodiment, a method for the production of a fiber is provided.The method comprises processing the concentrated aqueous silk fibroinsolution to form a fiber. Processing includes, for exampleelectrospinning or wet spinning. Alternatively, a fiber can be pulleddirectly from the solution. If desired, the fiber can be treated withmethanol, preferably by immersion, after processing. The fiber is thenpreferably washed with water.

A composition comprising a fiber that is produced by the method of thepresent invention and a therapeutic agent is also provided.

In another embodiment, a method of producing a silk foam is provided.The method comprises processing the concentrated aqueous silk solutionof the invention to produce a foam. Processing methods include, forexample, salt leaching, gas foaming, micropatterning, or by contactingsolution with a salt particle. The salt is preferably monovalent, e.g.NaCl, KCl, KFl, or NaBr. Alternatively, divalent salts, e.g. CaCl₂,MgSO₄, or MgCl₂, may also be used.

A composition comprising a foam produced by the method of the presentinvention and a therapeutic agent is also provided.

In another embodiment, a method of producing a film is provided. Themethod casting the concentrated aqueous salt solution to form a film. Incertain embodiments, it is useful to contact the film with water vapor.In addition, the film can be stretched mono-axially and biaxially.

A composition comprising a film that is produced by the method of thepresent invention and a therapeutic agent is also provided.

In another embodiment, a method of producing a silk hydrogel isprovided. The method comprises inducing a sol-gel transition in theconcentrated aqueous silk solution of the invention.

The sol-gel transition can be induced by an increase in the silk fibroinconcentration, an increase in temperature, a decrease in pH, an increasein the concentration of salt (e.g. KCl, NaCl, or CaCl₂.), or by additionof a polymer (e.g. polyethylene oxide (PEO).

A composition comprising a silk hydrogel that is produced by the methodof the present invention and a therapeutic agent is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the description, serve to explain the objects, advantages,and principles of the invention.

FIG. 1 illustrates one embodiment of the method of the present inventionto make highly concentrated regenerated silk fibroin solution.

FIG. 2 illustrates one embodiment of the method of the present inventionfor the preparation of porous silk fibroin scaffolds.

FIGS. 3 a and 3 b show (FIG. 3 a) X-ray diffraction and (FIG. 3 b) FTIRspectrum of a silk fibroin scaffold prepared by the water-based methoddescribed in Example II.

FIGS. 4 a and 4 b shown the mass of scaffolds remaining over time whenprepared from (FIG. 4 a) 4 or 8 wt % silk fibroin with NaCl of 850-1000μm diameter particle size, and (FIG. 4 b) of the scaffolds prepared with6 wt % prepared with various particle sizes of NaCl.

FIGS. 5 a and 5 b illustrate one embodiment of the present invention forsilk film preparation including (FIG. 5 a) water treatment and (FIG. 5b) stretching.

FIG. 6 shows the concentration of silk fibroin solution (filled symbol)and gel (open symbol) prepared by dialysis against PEG solutions(circle; 25 wt %, rectangle; 15 wt %, triangle; 10 wt %) at roomtemperature. Values are average±standard derivation of 3 samples.

FIG. 7 shows the gelation time of silk fibroin aqueous solutions atvarious temperatures (pH 6.5˜6.8, without ions). Values areaverage±standard derivation of 7 samples.

FIGS. 8 a, 8 b, and 8 c show the gelation time of silk fibroin aqueoussolutions with different Ca²⁺ (pH 5.6˜5.9) and K⁺ (pH 6.2˜6.4)concentrations at (FIG. 8 a) room temperature, (FIG. 8 b) 37° C. and(FIG. 8 c) 37° C. Values are average±standard derivation of 7 samples.

FIG. 9 shows the gelation time of silk fibroin aqueous solutions atvarious pHs (4 wt % silk fibroin; without ions; room temperature).Values are average±standard derivation of 7 samples.

FIG. 10 shows the gelation time of silk fibroin aqueous solutions atvarious PEO contents (4 wt % silk fibroin; pH 6.1˜6.4; without ions;room temperature). Values are average±standard derivation of 7 samples.

FIGS. 11 a and 11 b show the X-ray profiles of (FIG. 11 a) freeze-driedsilk fibroin solutions and (FIG. 11 b) hydrogels prepared from silkfibroin aqueous solution at 60° C.

FIGS. 12 a, 12 b, and 12 c show the compressive strength (FIG. 12 a),compressive modulus (FIG. 12 b) and strain at failure (FIG. 12 c) ofhydrogels prepared from silk fibroin aqueous solutions at varioustemperatures. **: Hydrogel prepared at 60° C. with the silk fibroinconcentration of 16 wt % was not crushed under the conditions used inthe study. Values are average±standard derivation of 5 samples.

DETAILED DESCRIPTION OF THE INVENTION

Methods for preparation of concentrated aqueous silk fibroin solutionsin the absence of organic solvents or harsh chemicals are described. Theprocess comprises forming a solution comprising silk fibroin.Preferably, the solution is in an aqueous salt, such as lithium bromide.The solution is then dialyzed against a hygroscopic polymer for asufficient time to result in an aqueous silk fibroin solution of between10-30 wt % or greater. A preferred hygroscopic polymer is polyethyleneglycol (PEG).

We have discovered that increasing the viscosity of the aqueous silkfibroin solution to at least 10 wt % allows for the formation of fibersby electrospinning, for the formation of porous 3-dimensional tissueengineering scaffolds, and for other applications, e.g., formation offoams and films, while avoiding the use of organic solvents that canpose problems when the processed materials are exposed to cells in vitroor in vivo. Dialysis of the solution against a hygroscopic polymer isalso sufficient to control water content in the formation of silkhydrogels.

As used herein, the term “fibroin” includes silkworm fibroin and insector spider silk protein (Lucas et al., Adv. Protein Chem 13: 107-242(1958)). Preferably, fibroin is obtained from a solution containing adissolved silkworm silk or spider silk. The silkworm silk protein isobtained, for example, from Bombyx mori, and the spider silk is obtainedfrom Nephila clavipes. In the alternative, the silk proteins suitablefor use in the present invention can be obtained from a solutioncontaining a genetically engineered silk, such as from bacteria, yeast,mammalian cells, transgenic animals or transgenic plants. See, forexample, WO 97/08315 and U.S. Pat. No. 5,245,012.

The silk fibroin solution to be concentrated can be prepared by anyconventional method known to one skilled in the art. For example, B.mori cocoons are boiled for about 30 minutes in an aqueous solution.Preferably, the aqueous solution is about 0.02M Na₂CO₃. The cocoons arerinsed, for example, with water to extract the sericin proteins and theextracted silk is dissolved in an aqueous salt solution. Salts usefulfor this purpose include, lithium bromide, lithium thiocyanate, calciumnitrate or other chemicals capable of solubilizing silk. Preferably, theextracted silk is dissolved in about 9-12 M LiBr solution. The salt isconsequently removed using, for example, dialysis.

The solution is then concentrated using, for example, dialysis against ahygroscopic polymer, for example, PEG, a polyethylene oxide, amylose orsericin.

Preferably, the PEG is of a molecular weight of 8,000-10,000 g/mol andhas a concentration of 25-50%. A slide-a-lyzer dialysis cassette(Pierce, MW CO 3500) is preferably used. However, any dialysis systemmay be used. The dialysis is for a time period sufficient to result in afinal concentration of aqueous silk solution between 10-30%. In mostcases dialysis for 2-12 hours is sufficient.

The concentrated aqueous solution of the present invention can beprocessed into hydrogels, foams, films, threads, fibers, meshes, andscaffolds using processes known in the art. See, e.g., Altman, et al.,Biomaterials 24:401, 2003.

Biocompatible polymers can be added to the silk solution to generatecomposite matrices in the process of the present invention.

Biocompatible polymers useful in the present invention include, forexample, polyethylene oxide (PEO) (U.S. Pat. No. 6,302,848),polyethylene glycol (PEG) (U.S. Pat. No. 6,395,734), collagen (U.S. Pat.No. 6,127,143), fibronectin (U.S. Pat. No. 5,263,992), keratin (U.S.Pat. No. 6,379,690), polyaspartic acid (U.S. Pat. No. 5,015,476),polylysine (U.S. Pat. No. 4,806,355), alginate (U.S. Pat. No.6,372,244), chitosan (U.S. Pat. No. 6,310,188), chitin (U.S. Pat. No.5,093,489), hyaluronic acid (U.S. Pat. No. 387,413), pectin (U.S. Pat.No. 6,325,810), polycaprolactone (U.S. Pat. No. 6,337,198), polylacticacid (U.S. Pat. No. 6,267,776), polyglycolic acid (U.S. Pat. No.5,576,881), polyhydroxyalkanoates (U.S. Pat. No. 6,245,537), dextrans(U.S. Pat. No. 5,902,800), and polyanhydrides (U.S. Pat. No. 5,270,419).Two or more biocompatible polymers can be used.

Silk films can be produced by preparing the concentrated aqueous silkfibroin solution and casting the solution. In one embodiment, the filmis contacted with water or water vapor, in the absence of alcohol. Thefilm can then be drawn or stretched mono-axially or biaxially. See, forexample, FIGS. 5 a and 5 b. The stretching of a silk blend film inducesmolecular alignment of the film and thereby improves the mechanicalproperties of the film.

In one embodiment, the film comprises from about 50 to about 99.99 partby volume aqueous silk protein solution and from about 0.01 to about 50part by volume biocompatible polymer e.g., polyethylene oxide (PEO).Preferably, the resulting silk blend film is from about 60 to about 240μm thick, however, thicker samples can easily be formed by using largervolumes or by depositing multiple layers.

Foams may be made from methods known in the art, including, for example,freeze—drying and gas foaming in which water is the solvent or nitrogenor other gas is the blowing agent, respectively. Alternately the foam ismade by contacting the silk fibroin solution with granular salt. Thepore size of foams can be controlled, for example by adjusting theconcentration of silk fibroin and the particle size of a granular salt(for example, the preferred diameter of the salt particle is betweenabout 50 microns and about 1000 microns). The salts can be monovalent ordivalent. Preferred salts are monovalent, such as NaCl and KCl. Divalentsalts, such as CaCl₂ can also be used. Contacting the concentrated silkfibroin solution with salt is sufficient to induce a conformationalchange of the amorphous silk to a β-sheet structure that is insoluble inthe solution. After formation of the foam, the excess salt is thenextracted, for example, by immersing in water. The resultant porous foamcan then be dried and the foam can be used, for example, as a cellscaffold in biomedical application. See, FIG. 2.

In one embodiment, the foam is a micropatterned foam. Micropatternedfoams can be prepared using, for example, the method set forth in U.S.Pat. No. 6,423,252, the disclosure of which is incorporated herein byreference. The method comprises contacting the concentrated silksolution of the present invention with a surface of a mold, the moldcomprising on at least one surface thereof a three-dimensional negativeconfiguration of a predetermined micropattern to be disposed on andintegral with at least one surface of the foam, lyophilizing thesolution while in contact with the micropatterned surface of the mold,thereby providing a lyophilized, micropatterned foam, and removing thelyophilized, micropatterned foam from the mold. Foams prepared accordingthis method comprise a predetermined and designed micropattern on atleast one surface, which pattern is effective to facilitate tissuerepair, ingrowth or regeneration.

Fibers may be produced using, for example, wet spinning orelectrospinning. Alternatively, as the concentrated solution has agel-like consistency, a fiber can be pulled directly from the solution.

Electrospinning can be performed by any means known in the art (see, forexample, U.S. Pat. No. 6,110,590). Preferably, a steel capillary tubewith a 1.0 mm internal diameter tip is mounted on an adjustable,electrically insulated stand. Preferably, the capillary tube ismaintained at a high electric potential and mounted in the parallelplate geometry. The capillary tube is preferably connected to a syringefilled with silk solution. Preferably, a constant volume flow rate ismaintained using a syringe pump, set to keep the solution at the tip ofthe tube without dripping. The electric potential, solution flow rate,and the distance between the capillary tip and the collection screen areadjusted so that a stable jet is obtained. Dry or wet fibers arecollected by varying the distance between the capillary tip and thecollection screen.

A collection screen suitable for collecting silk fibers can be a wiremesh, a polymeric mesh, or a water bath. Alternatively and preferably,the collection screen is an aluminum foil. The aluminum foil can becoated with Teflon fluid to make peeling off the silk fibers easier. Oneskilled in the art will be able to readily select other means ofcollecting the fiber solution as it travels through the electric field.As is described in more detail in the Examples section below, theelectric potential difference between the capillary tip and the aluminumfoil counter electrode is, preferably, gradually increased to about 12kV, however, one skilled in the art should be able to adjust theelectric potential to achieve suitable jet stream.

The present invention additionally provides a non-woven network offibers comprising a fiber of the present invention. The fiber may alsobe formed into yarns and fabrics including for example, woven or weavedfabrics.

The fibroin silk solution of the present invention may also be coatedonto various shaped articles including biomedical devices (e.g. stents),and silk or other fibers, including fragments of such fibers.

Silk hydrogels can be prepared by methods known in the art, and asexemplified herein. The sol-gel transition of the concentrated silkfibroin solution can be modified by changes in silk fibroinconcentration, temperature, salt concentrations (e.g. CaCl₂, NaCl, andKCl), pH, hydrophilic polymers, and the like. Before the sol-geltransition, the concentrated aqueous silk solution can be placed in amold or form. The resulting hydrogel can then be cut into any shape,using, for example a laser.

The materials produced using the present invention, e.g., hydrogels,fibers, films, foams, or meshes, may be used in a variety of medicalapplications such as a drug (e.g, small molecule, protein, or nucleicacid) delivery device, including controlled release systems, woundclosure systems, including vascular wound repair devices, hemostaticdressings, patches and glues, sutures, and in tissue engineeringapplications, such as, for example, scaffolds for tissue regeneration,ligament prosthetic devices and in products for long-term orbio-degradable implantation into the human body. Films may also be usedfor a wide range of materials science and engineering needs, such ascontrolled drug release systems, coatings, composites or as stand alonematerials.

Additionally, these biomaterials can be used for organ repairreplacement or regeneration strategies that may benefit from theseunique scaffolds, including but are not limited to, spine disc, cranialtissue, dura, nerve tissue, liver, pancreas, kidney, bladder, spleen,cardiac muscle, skeletal muscle, tendons, ligaments and breast tissues.

In another embodiment of the present invention, silk biomaterials cancontain therapeutic agents. To form these materials, the silk solutionis mixed with a therapeutic agent prior to forming the material orloaded into the material after it is formed. The variety of differenttherapeutic agents that can be used in conjunction with the biomaterialsof the present invention is vast and includes small molecules, proteins,peptides and nucleic acids. In general, therapeutic agents which may beadministered via the invention include, without limitation:antiinfectives such as antibiotics and antiviral agents;chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents;analgesics and analgesic combinations; anti-inflammatory agents;hormones such as steroids; growth factors (bone morphogenic proteins(i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GFD-5, GFD-7 andGFD-8), epidermal growth factor (EGF), fibroblast growth factor (i.e.FGF 1-9), platelet derived growth factor (PDGF), insulin like growthfactor (IGF-I and IGF-II), transforming growth factors (i.e. TGF-β-III),vascular endothelial growth factor (VEGF)); anti-angiogenic proteinssuch as endostatin, and other naturally derived or geneticallyengineered proteins, polysaccharides, glycoproteins, or lipoproteins.Growth factors are described in The Cellular and Molecular Basis of BoneFormation and Repair by Vicki Rosen and R. Scott Thies, published by R.G. Landes Company, hereby incorporated herein by reference.Additionally, the silk biomaterials of the present invention can be usedto deliver any type of molecular compound, such as, pharmacologicalmaterials, vitamins, sedatives, steroids, hypnotics, antibiotics,chemotherapeutic agents, prostaglandins, and radiopharmaceuticals. Thedelivery system of the present invention is suitable for delivery theabove materials and others including but not limited to proteins,peptides, nucleotides, carbohydrates, simple sugars, cells, genes,anti-thrombotics, anti-metabolics, growth factor inhibitor, growthpromoters, anticoagulants, antimitotics, fibrinolytics,anti-inflammatory steroids, and monoclonal antibodies.

Silk biomaterials containing bioactive materials may be formulated bymixing one or more therapeutic agents with the polymer used to make thematerial. Alternatively, a therapeutic agent could be coated on to thematerial preferably with a pharmaceutically acceptable carrier. Anypharmaceutical carrier can be used that does not dissolve the silkmaterial. The therapeutic agents, may be present as a liquid, a finelydivided solid, or any other appropriate physical form.

The biomaterials described herein can be further modified afterfabrication. For example, the scaffolds can be coated with additives,such as bioactive substances that function as receptors orchemoattractors for a desired population of cells. The coating can beapplied through absorption or chemical bonding.

Additives suitable for use with the present invention includesbiologically or pharmaceutically active compounds. Examples ofbiologically active compounds include, but are not limited to: cellattachment mediators, such as collagen, elastin, fibronectin,vitronectin, laminin, proteoglycans, or peptides containing knownintegrin binding domains e.g. “RGD” integrin binding sequence, orvariations thereof, that are known to affect cellular attachment(Schaffner P & Dard 2003 Cell Mol Life Sci. January; 60(1):119-32;Hersel U. et al. 2003 Biomaterials. November; 24(24):4385-415);biologically active ligands; and substances that enhance or excludeparticular varieties of cellular or tissue ingrowth. For example, thesteps of cellular repopulation of a 3-dimensional scaffold matrixpreferably are conducted in the presence of growth factors effective topromote proliferation of the cultured cells employed to repopulate thematrix. Agents that promote proliferation will be dependent on the celltype employed. For example, when fibroblast cells are employed, a growthfactor for use herein may be fibroblast growth factor (FGF), mostpreferably basic fibroblast growth factor (bFGF) (Human RecombinantbFGF, UPSTATE Biotechnology, Inc.). Other examples of additive agentsthat enhance proliferation or differentiation include, but are notlimited to, osteoinductive substances, such as bone morphogenic proteins(BMP); cytokines, growth factors such as epidermal growth factor (EGF),platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-Iand II) TGF-β, and the like. As used herein, the term additive alsoencompasses antibodies, DNA, RNA, modified RNA/protein composites,glycogens or other sugars, and alcohols.

The biomaterials can be shaped into articles for tissue engineering andtissue guided regeneration applications, including reconstructivesurgery. The structure of the scaffold allows generous cellularingrowth, eliminating the need for cellular preseeding. The scaffoldsmay also be molded to form external scaffolding for the support of invitro culturing of cells for the creation of external support organs.

The scaffold functions to mimic the extracellular matrices (ECM) of thebody. The scaffold serves as both a physical support and an adhesivesubstrate for isolated cells during in vitro culture and subsequentimplantation. As the transplanted cell populations grow and the cellsfunction normally, they begin to secrete their own ECM support.

In the reconstruction of structural tissues like cartilage and bone,tissue shape is integral to function, requiring the molding of thescaffold into articles of varying thickness and shape. Any crevices,apertures or refinements desired in the three-dimensional structure canbe created by removing portions of the matrix with scissors, a scalpel,a laser beam or any other cutting instrument. Scaffold applicationsinclude the regeneration of tissues such as nervous, musculoskeletal,cartilaginous, tendenous, hepatic, pancreatic, ocular, integumenary,arteriovenous, urinary or any other tissue forming solid or holloworgans.

The scaffold may also be used in transplantation as a matrix fordissociated cells, e.g., chondrocytes or hepatocytes, to create athree-dimensional tissue or organ. Tissues or organs can be produced bymethods of the present invention for any species.

A number of different cell types or combinations thereof may be employedin the present invention, depending upon the intended function of thetissue engineered construct being produced. These cell types include,but are not limited to: smooth muscle cells, skeletal muscle cells,cardiac muscle cells, epithelial cells, endothelial cells, urothelialcells, fibroblasts, myoblasts, chondrocytes, chondroblasts, osteoblasts,osteoclasts, keratinocytes, hepatocytes, bile duct cells, pancreaticislet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary,ovarian, testicular, salivary gland cells, adipocytes, and precursorcells. For example, smooth muscle cells and endothelial cells may beemployed for muscular, tubular constructs, e.g., constructs intended asvascular, esophageal, intestinal, rectal, or ureteral constructs;chondrocytes may be employed in cartilaginous constructs; cardiac musclecells may be employed in heart constructs; hepatocytes and bile ductcells may be employed in liver constructs; epithelial, endothelial,fibroblast, and nerve cells may be employed in constructs intended tofunction as replacements or enhancements for any of the wide variety oftissue types that contain these cells. In general, any cells may beemployed that are found in the natural tissue to which the construct isintended to correspond. In addition, progenitor cells, such as myoblastsor stem cells, may be employed to produce their correspondingdifferentiated cell types. In some instances it may be preferred to useneonatal cells or tumor cells.

Cells can be obtained from donors (allogenic) or from recipients(autologous). Cells can also be of established cell culture lines, oreven cells that have undergone genetic engineering. Pieces of tissue canalso be used, which may provide a number of different cell types in thesame structure.

Appropriate growth conditions for mammalian cells are well known in theart (Freshney, R. I. (2000) Culture of Animal Cells, a Manual of BasicTechnique. Hoboken N.J., John Wiley & Sons; Lanza et al. Principles ofTissue Engineering, Academic Press; 2nd edition May 15, 2000; and Lanza& Atala, Methods of Tissue Engineering Academic Press; 1st editionOctober 2001). Cell culture media generally include essential nutrientsand, optionally, additional elements such as growth factors, salts,minerals, vitamins, etc., that may be selected according to the celltype(s) being cultured. Particular ingredients may be selected toenhance cell growth, differentiation, secretion of specific proteins,etc. In general, standard growth media include Dulbecco's Modified EagleMedium, low glucose (DMEM), with 110 mg/L pyruvate and glutamine,supplemented with 10-20% fetal bovine serum (FBS) or calf serum and 100U/ml penicillin are appropriate as are various other standard media wellknown to those in the art. Growth conditions will vary dependent on thetype of mammalian cells in use and tissue desired.

In one embodiment, methods are provided for producing bone or cartilagetissue in vitro comprising culturing multipotent cells on a porous silkfibroin scaffold under conditions appropriate for inducing bone orcartilage formation. Suitable conditions for the generation of bone andcartilage are well known to those skilled in the art. For example,conditions for the growth of cartilage tissue often comprisenonessential amino acids, ascorbic acid-2-phosphate, dexamethasone,insulin, and TGF-β1. In one preferred embodiment, the nonessential aminoacids are present at a concentration of 0.1 mM, ascorbicacid-2-phosphate is present at a concentration of 50 ug/ml,dexamethasone is present at a concentration of 10 nM, insulin is presentat a concentration of 5 ug/ml and TGF-β1 is present at a concentrationof 5 ng/ml. Suitable conditions for the growth of bone often includeascorbic acid-2-phosphate, dexamethasone, β-glycerolphoasphate andBMP-2. In a preferred embodiment, ascorbic acid-2-phosphate is presentat a concentration of 50 ug/ml, dexamethasone is present at aconcentration of 10 nM, β-glycerolphoasphate is present at aconcentration of 7 mM and BMP-2 is present at a concentration of 1ug/ml.

In general, the length of the growth period will depend on theparticular tissue engineered construct being produced. The growth periodcan be continued until the construct has attained desired properties,e.g., until the construct has reached a particular thickness, size,strength, composition of proteinaceous components, and/or a particularcell density. Methods for assessing these parameters are known to thoseskilled in the art.

Following a first growth period the construct can be seeded with asecond population of cells, which may comprise cells of the same type asused in the first seeding or cells of a different type. The constructcan then be maintained for a second growth period which may be differentin length from the first growth period and may employ different growthconditions. Multiple rounds of cell seeding with intervening growthperiods may be employed.

In one preferred embodiment, tissues and organs are generated forhumans. In other embodiments, tissues and organs are generated foranimals such as, dogs, cats, horses, monkeys, or any other mammal.

The cells are obtained from any suitable donor, either human or animal,or from the subject into which they are to be implanted. As used herein,the term “host” or “subject” includes mammalian species, including, butnot limited to, humans, monkeys, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats.

The cells that are used for methods of the present invention should bederived from a source that is compatible with the intended recipient.The cells are dissociated using standard techniques and seeded onto andinto the scaffold. In vitro culturing optionally may be performed priorto implantation. Alternatively, the scaffold is implanted into thesubject, allowed to vascularize, then cells are injected into thescaffold. Methods and reagents for culturing cells in vitro andimplantation of a tissue scaffold are known to those skilled in the art.

Cells can be seeded within the matrix either pre- or post matrixformation, depending on the method of matrix formation. Uniform seedingis preferable. In theory, the number of cells seeded does not limit thefinal tissue produced, however optimal seeding may increase the rate ofgeneration. The number of seeded cells can be optimized using dynamicseeding (Vunjak-Novakovic et al. Biotechnology Progress 1998; Radisic etal. Biotechnoloy and Bioengineering 2003).

It is another aspect of the invention that the 3-dimensional porous silkscaffold, described herein, can itself be implanted in vivo and serve astissue substitute (e.g. to substitute for bone or cartilage). Suchimplants, would require no seeding of cells, but contain an additione.g., RGD, that attracts cells.

In one embodiment, silk matrix scaffolds are seeded with multipotentcells in the presence of media that induces either bone or cartilageformation. Suitable media for the production of cartilage and bone arewell known to those skilled in the art.

As used herein, “multipotent” cells have the ability to differentiateinto more than one cell type in response to distinct differentiationsignals. Examples of multipotent cells include, but are not limited to,bone marrow stromal cells (BMSC) and adult or embryonic stem cells. In apreferred embodiment BMSCs are used. BMSCs are multipotential cells ofthe bone marrow which can proliferate in an undifferentiated state andwith the appropriate extrinsic signals, differentiate into cells ofmesenchymal lineage, such as cartilage, bone, or fat (Friedenstein, A.J. 1976. Int Rev Cytol 47:327-359; Friedenstein et al. 1987. Cell TissueKinet 20:263-272; Caplan, A. I. 1994. Clin Plast Surg 21:429-435; Mackayet al. 1998. Tissue Eng 4:415-428; Herzog et al. Blood. 2003 Nov. 15;102(10):3483-93. Epub 2003 Jul. 31).

The formation of cartilaginous tissue or bone can be monitored by assayswell known to those in the art including, but not limited to, histology,immunohistochemistry, and confocal or scanning electron microscopy (Holyet al., J. Biomed. Mater. Res (2003) 65A:447-453).

Using silk based scaffolds, organized tissue with a predetermined formand structure can be produced either in vitro or in vivo. For example,tissue that is produced ex vivo is functional from the start and can beused as an in vivo implant. Alternatively, the silk based structure canbe seeded with cells capable of forming either bone or cartilage andthen implanted as to promote growth in vivo. Thus, the scaffolds can bedesigned to form tissue with a “customized fit” that is specificallydesigned for implantation in a particular patient. For example,cartilaginous tissue or bone tissue produced by methods of the presentinvention can be used to replace large cartilage or bone defects foundin musculoskeletal disorders and degenerative diseases such asosteoarthritis or rheumatism. Engineered bone and cartilage are alsosuitable for spine and joint replacements such as, elbow, knee, hip orfinger joints or can be used in osteochondral implants.

All biomaterials of the present intention may be sterilized usingconventional sterilization process such as radiation based sterilization(i.e. gamma-ray), chemical based sterilization (ethylene oxide),autoclaving, or other appropriate procedures. Preferably thesterilization process will be with ethylene oxide at a temperaturebetween 52-55° C. for a time of 8 hours or less. After sterilization thebiomaterials may be packaged in an appropriate sterilize moistureresistant package for shipment and use in hospitals and other healthcare facilities.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of theinvention, the preferred methods and materials are described below. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference. In addition, thematerials, methods and examples are illustrative only and not intendedto be limiting. In case of conflict, the present specification,including definitions, controls.

The invention will be further characterized by the following exampleswhich are intended to be exemplary of the invention.

EXAMPLES Example I Preparation of Pure Silk Fibers from Water fromRegenerated Silk Solution by Electrospinning Methods

Preparation of a Regenerated B. mori Silk Fibroin Solution

B. mori silk fibroin was prepared as follows as a modification of ourearlier procedure (Sofia, et al., Journal of Biomedical MaterialsResearch 2001, 54, 139-148). Cocoons were boiled for 30 min in anaqueous solution of 0.02 M Na₂CO₃, then rinsed thoroughly with water toextract the glue-like sericin proteins. The extracted silk was thendissolved in 9.3 M LiBr solution at room temperature yielding a 20%(w/v) solution. This solution was dialyzed in water using aSlide-a-Lyzer dialysis cassette (Pierce, MWCO 2000) for 48 hours. Thefinal concentration of aqueous silk solution was 8.0 wt %, which wasdetermined by weighing the remaining solid after drying.

This solution was concentrated further by exposure to an aqueouspolyethylene glycol (PEG) (MW 8,000 to 10,000) solution (25-50 wt %) onthe outside of a Slide-a-Lyzer dialysis cassette (Pierce, MWCO 3500) for2 to 12 hrs by osmotic pressure (FIG. 1). The final concentration ofaqueous silk solution could be formed to between 10-30 wt % or greater.

Electrospinning

In order to increase the viscosity of aqueous silk solution above 8 wt %for spinning, the solution was concentrated using the PEG solutionmethod as described above. This was required since the viscosity andsurface tension of the pure silk solution (8 wt %) was not high enoughto maintain a stable drop at the end of the capillary tip. The increaseof silk solutions generated a viscosity and surface tension suitable forelectrospinning With the new more concentrated pure silk solutions(10-30%), direct spinning is now feasible. The distance between the tipand the collector was 10-15 cm and flow rate of the fluid was 0.01 to0.05 ml/min. As the potential difference between the capillary tip andthe aluminum foil counter electrode was gradually increased 30 kV (E=2-3kV/cm), the drop at the end of the capillary tip elongated from ahemispherical shape into a cone shape. The morphology and diameters ofthe electrospun fibers were examined using SEM. Silk/PEO blend solutionproduced microsize fibers with diameters 1.5 μm to 25 μm. The morphologyof fiber surface and fracture surface in liquid nitrogen was wellmatched with native silk fiber.

Example II Preparation of Silk Fibroin Scaffolds

Porous three-dimensional scaffolds were prepared from silk fibroinaqueous solutions by salt-leaching. By adjusting the concentration ofsilk fibroin and the particle size of granular NaCl, the morphologicaland functional properties of the scaffolds could be controlled. Thescaffolds had highly homogeneous and interconnected pores and showedpore sizes ranging from 470 to 940 um depending on the mode ofpreparation. The scaffolds had porosities >90%. The compressive strengthand modulus of scaffolds was up to 320±10 KPa and 3330±500 KPa,respectively. The scaffolds were fully degraded by protease during 21days. These new silk-based 3-D matrices provide useful properties asbiomaterial matrices for tissue engineering due to the all-aqueous modeof preparation, control of pore size, connectivity of pores,degradability and useful mechanical features.

Methods Preparation of Silk Fibroin Aqueous Solution

Cocoons of B. mori were boiled for 20 min in an aqueous solution of 0.02M Na₂CO₃, and then rinsed thoroughly with distilled water to extract theglue-like sericin proteins and wax. The extracted silk fibroin was thendissolved in 9.3 M LiBr solution at 60° C. for 4 hrs, yielding a 20 w/v% solution. This solution was dialyzed in distilled water using aSlide-a-Lyzer dialysis cassette (MWCO 3500, Pierce) for 2 days. Thefinal concentration of silk fibroin aqueous solution was ca. 8 w/v %,which was determined by weighing the remaining solid after drying. Toprepare concentrated silk fibroin solution, 10 ml of 8 w/v % silkfibroin solution was dialyzed against 1 liter of 25 wt % polyethyleneglycol (PEG, 10,000 g/mol) solution at room temperature by usingSlide-a-Lyzer dialysis cassettes (MWCO 3500). After the required time,the concentrated silk fibroin solution was slowly collected by syringeto avoid excessive shearing and the concentration was determined. Silkfibroin aqueous solutions with concentration less than 8 wt % wereprepared by diluting with distilled water. All solutions were stored at7° C. before use to avoid premature precipitation. Silk fibroin filmsprepared from 8 w/v % solutions were evaluated to verify the removal ofLi⁺ ion by XPS; no residual Li⁺ ion was detected.

Preparation of Silk Fibroin Scaffolds

Four grams of granular NaCl (particle size; 300˜1180 um) were added to 2ml of silk fibroin aqueous solution (4˜10 wt %) in disk-shaped Tefloncontainers (FIG. 2 a). The container was covered and left at roomtemperature. After 24 hrs, the container was immersed in water and theNaCl was extracted for 2 days. The porous silk fibroin scaffolds formedin this process were stored in water at 7° C. before use.

X-ray Diffraction

X-ray diffraction of freeze-dried samples of the scaffold were obtainedwith Ni-filtered Cu—Kα radiation (λ=0.15418 nm) from a Rigaku RU-200BHrotating-anode X-ray generator operating at 40 kV and 40 mA. X-raydiffraction patterns were recorded with a point collimated beam and aimaging plate (Fuji Film BAS-IP SR 127) in an evacuated camera. Thecamera length was calibrated with NaF (d=0.23166 nm).

FTIR Spectroscopy

Approximately 1 mg of freeze-dried sample was pressed into a pellet with200 mg of potassium bromide and Fourier transform infrared (FTIR)spectrum was recorded with an accumulation of 64 scans and a resolutionof 4 cm-1 by Nicolet Magna 860.

Scanning Electron Microscopy (SEM)

Silk scaffolds were cut into sections in distilled water using a razorblade and then freeze-dried. Samples were sputter coated with gold. Themorphology of scaffolds was observed with a LEO Gemini 982 FieldEmission Gun SEM. Pore size was obtained using ImageJ software developedat the US National Institutes of Health.

Porosity

The density and porosity of the silk scaffolds were measured by liquiddisplacement (Zhang, R. Y., et al., J. Biomed. Mater. Res. 1999, 44,446-455). Hexane was used as the displacement liquid as it permeatesthrough silk scaffolds without swelling or shrinking the matrix. Thesilk scaffold (dry weight, W) was immersed in a known volume (V1) ofhexane in a graduated cylinder for 5 min. The total volume of hexane andthe hexane-impregnated scaffold was recorded as V2. Thehexane-impregnated scaffold was then removed from the cylinder and theresidual hexane volume was recorded as V3. The total volume of thescaffold was:

V=(V2−V1)+(V1−V3)=V2−V3.

-   -   V2−V1 is the volume of the polymer scaffold and V1−V3 is the        volume of hexane within the scaffold. The porosity of the        scaffold (ε) was obtained by:

ε(%)=(V1−V3)/(V2−V3)×100

Swelling Properties

Silk fibroin scaffolds were immersed in distilled water at roomtemperature for 24 hrs. After excess water was removed, the wet weightof the scaffold (Ws) was determined. Samples were then dried in an ovenat 65° C. under vacuum overnight and the dry weight of scaffolds (Wd)was determined. The swelling ratio of the scaffold and the water contentin the scaffold were calculated as follows:

Swelling ratio=(Ws−Wd)/Wd

Water uptake(%)=[(Ws−Wd)/Ws]×100

Mechanical Properties

Resistance to mechanical compression of the scaffolds (12 mm diameter,10 mm height, disks) were performed on an Instron 8511 equipped with a0.1 KN load cell at room temperature. The crosshead speed was 10 mm/min.The compression tests were conducted conventionally as anopen-sided/confined method. Four samples were evaluated for eachcomposition. Cylinder-shaped samples measuring 12 mm in diameter and 10mm in height were used, according to a modification based on the ASTMmethod F451-95. The compressive stress and strain were graphed and theaverage compressive strength as well as the compressive modulus andstandard deviation determined. The elastic modulus was defined by theslope of the initial linear section of the stress-strain curve. Thecompressive strength was determined by drawing a line parallel to this,starting at 1% strain. The point at which this line crossed thestress-strain curve was defined as the compressive strength of the foam(Thomson R C et al., Biomaterials 1998, 19; 1935-1943).

In Vitro Enzymatic Degradation

The degradation of the silk fibroin scaffolds was evaluated usingprotease XIV (EC 3.4.24.31, Sigma-Aldrich) with an activity of 5.6 U/mg.Samples (12 mm diameter, 5 mm height) were immersed in 5 ml of phosphatebuffer saline (pH 7.4) containing protease (1 U) at 37° C. After thespecific time samples were washed with phosphate buffer saline anddistilled water, and freeze-dried. The enzyme solution was replaced withnewly prepared solution every 24 hrs. For controls, samples wereimmersed in phosphate buffer saline without enzyme.

Results and Discussion Preparation of Water-Based Scaffolds

Porous silk fibroin scaffolds were prepared using a salt-leaching methodthat has been previously used in the preparation of porous scaffoldsfrom other polymers such as collagen and polylactic acid. The pore sizeand the porosity of the scaffolds were regulated by the addition ofgranular NaCl with particle sizes of diameter 300 to 1180 μm to the silkfibroin aqueous solution. In this process, some of the surface of theNaCl particles dissolved in the silk fibroin aqueous solution, whilemost of the salt was retained as solid particles because of saturationof the solution. The silk fibroin aqueous solutions formed intohydrogels in the mixture after ˜24 hrs, which resulted in the formationof water-stable porous matrices. Table 1 shows the silk fibroinconcentrations and particle sizes of NaCl used in the study. With anincrease in silk fibroin concentration, matrices were homogeneouslyformed through the use of larger particle sizes of the NaCl. When NaClwith particle sizes of 500 to 600 μm were added to 8 wt % silk fibroinsolution, the surface of the silk fibroin aqueous solutions rapidlyformed a hydrogel.

TABLE 1 Preparation of scaffolds from various silk fibroinconcentrations and particle sizes of NaCl. Particle size of sodium Silkfibroin concentration (w/v %) chloride (μm) 4 6 8 10 1000~1180 ∘ ∘ ∘ ∘ 850~1000 ∘ ∘ ∘ Δ 710~850 ∘ ∘ ∘ x 600~700 ∘ ∘ Δ x 500~600 ∘ Δ x x425~500 Δ x x x 300~425 x x x x degree of homogeneity: ∘ > Δ > x

In concentrated salt solutions, solvating forces are significantlyaltered from those in dilute electrolyte solutions because salt ionschange the structure of the intervening water (Curtis R A et al.,Biophys Chem 2002, 98:249-265). The effect of concentrated saltsolutions with chloride ion, such as NaCl, KCl, CaCl₂ and MgCl₂, on silkfibroin was determined at salt concentrations up to 3 M at roomtemperature. When a drop of silk fibroin solution (8 wt %) was added toconcentrated salt solutions of 3 M, silk hydrogels formed immediately inthe NaCl and KCl solutions but not in the CaCl₂ and MgCl₂ solutions.Ions are classified as kosmotropic or chaotropic, based on their sizeand charge (Grigsby J J et al., Biophys Chem 2001, 91:231-243). Ionswith high charge density such as Ca²⁺ and Mg²⁺ are highly kosmotropic,and ions with low charge density such as K⁺ are chaotropic. Na⁺ isweakly kosmotropic and Cl⁻ is weakly chaotropic. Kosmotropic ions bindadjacent water molecules more strongly than chaotropic ions. Inaddition, kosmotropic ions strongly interact with oppositely chargedresidues on the protein surface due to their high charge density. At lowsalt concentration, the solution contains a sufficient number of watermolecules to hydrate both the protein surface and the ions. At highersalt concentrations, more water molecules are needed to hydrate theincreasing number of ions. Therefore water molecules are easily removedfrom the proteins as concentrations of salt solutions increase.

From the primary sequence of the silkworm silk fibroin heavy chain,seven internal hydrophobic blocks and seven much smaller internalhydrophilic blocks, with two large hydrophilic blocks at the chain endsare present (Zhou, C. Z., et al., Nucleic Acids Res. 2000, 28,2413-2419). The percentage of hydrophobic residues in silk fibroin is79% (Braun, F. N., et al., Int. J. Biol. Macromol. 2003, 32, 59-65) andthe repetitive sequence in these hydrophobic blocks consists of GAGAGS(SEQ ID NO: 4) peptides that dominate the β-sheet structure that formsthe crystalline regions in silk fibroin fibers and films (Mita, K., etal., J. Mol. Evol. 1994, 38, 583-592).

Since protein solubility typically decreases as salt concentrationrises, interactions between proteins become favored (Curtis, R. A., etal., Biophys. Chem. 2002, 98, 249-265). It is well known that thehydrophobic interactions between non-polar residues increase withaddition of salt, leading to the salting-out effect (Robinson, D. R., etal., J. Am. Chem. Soc. 1965, 87, 2470-2479). The behavior of the fibroinin the salt system described may be related to the role of the salt ionsin extracting water that would otherwise coat the hydrophobic fibroindomains, promoting chain-chain interactions leading to the new morestable structure. These hydrophobic interactions induce protein folding,resulting in β-sheet formation (Li, G. Y., et al., Biochem. 2001, 268,6600-6606).

Alginate or glass beads were examined to further clarify the ion effectson hydrogelation of silk fibroin (8 wt %). While gelation time of silkfibroin with glass beads showed a similar result as that observed over30 days with silk fibroin in a previous study (Kim U J et al.,Biomacromolecules, in press), the gelation time of the silk fibroinsolution with alginate beads was ˜2 times faster due, presumably due tothe removal of water molecules from the proteins associated with theswelling of the alginate beads. Compared with the gelation time (24 hrs)of silk fibroin in saturated NaCl solution, salt ions strongly inducedprotein-protein interactions.

Structural Analysis

Structural changes in the silk fibroin were determined by X-raydiffraction and FTIR (FIG. 3). X-ray diffraction of silk fibroinscaffolds showed a distinct peak at 20.8° and a minor peak and 24.6°.These peaks were almost the same as those of the β-sheet crystallinestructure (silk II) of native silk fibroin (Asakura, T., et al.,Macromolecules 1985, 18, 1841-1845). The results indicate aβ-crystalline spacing distances of 4.3 and 3.6 Å according to the 20.8°and 24.6° reflections, respectively. FTIR spectra of silk fibroinscaffolds showed characteristic peaks of silk II at 1701 cm⁻¹ and 1623cm⁻¹ (amide I) (Asakura, T., et al., Macromolecules 1985, 18,1841-1845). Silk fibroin in aqueous solution at neutral pH exhibited arandom coil conformation. From the results of the X-ray diffraction andFTIR analyses, the formation of silk fibroin scaffolds from thesesolutions induced a conformational transition from random coil toβ-sheet.

Morphology

SEM images of freeze-dried scaffolds prepared from various silk fibroinconcentrations and various sized particles of NaCl showed highlyinterconnected porous structures and the pore distribution washomogeneous in the whole scaffolds except for a thin layer formed on thetop surface of the scaffolds, the air-water interface. The scaffoldsshowed rough pore surfaces highly interconnected by a number of smallerpores. Globular-like structures, 1˜3 μm in diameter, were observed onthe surfaces of the pores. With an increase in silk fibroinconcentration, the pore walls were thicker. Table 2 shows actual poresizes in the scaffolds, ranging from 350 to 920 μm.

TABLE 2 Measured pore sizes (μm) of silk fibroin scaffolds. Particlesize of sodium Silk fibroin concentration (w/v %) chloride (μm) 4 6 8 101000~1180 940 ± 50 930 ± 40 920 ± 50 920 ± 50  850~1000 760 ± 30 750 ±50 750 ± 20 — 710~850 650 ± 30 650 ± 50 640 ± 30 — 600~700 570 ± 30 550± 30 — — 500~600 470 ± 30 — — — Values are average ± standard derivation(N = 20).

The actual pore sizes in the scaffolds were 8090% smaller than theparticle size of NaCl used in the process. The pore sizes in scaffoldsprepared with the same particle size of NaCl, regardless of theconcentration of silk fibroin used, resulted in similar sized pores.

Porosity and Swelling Properties

Silk fibroin scaffolds with >90% porosity were formed and porositiesincreased with a decrease in pore size and silk fibroin concentrations(Table 3). These values were similar as those (84˜98%) of HFIP-derivedsilk scaffolds prepared by salt leaching or gas forming (Nazarov R, etal., Biomacromolecules, in press). Swelling ratio and water uptake ofthe scaffolds are shown in Tables 4 and 5.

TABLE 3 Porosity (%) of silk fibroin scaffolds Particle size of sodiumSilk fibroin concentration (w/v %) chloride (μm) 4 6 8 10 1000~1180 95 ±1.8 93 ± 0.7 92 ± 1.3 85 ± 1.5  850~1000 95 ± 1.5 95 ± 0.2 94 ± 0.2 —710~850 97 ± 0.4 96 ± 1.6 95 ± 1.5 — 600~700 97 ± 1.6 97 ± 0.6 — —500~600 97 ± 0.5 — — — Values are average ± standard derivation (N = 3).

TABLE 4 Swelling ratio of silk fibroin scaffolds. Particle size ofsodium Silk fibroin concentration (w/v %) chloride (μm) 4 6 8 101000~1180 55.3 ± 3.8 36.1 ± 0.1 23.6 ± 1.2 19.2 ± 4.3  850~1000 50.0 ±0.2 29.8 ± 0.6 21.5 ± 1.9 — 710~850 48.6 ± 2.0 28.9 ± 1.5 19.8 ± 0.2 —600~700 46.8 ± 2.6 28.4 ± 2.7 — — 500~600 47.6 ± 2.1 — — — Values areaverage ± standard derivation (N = 3).

TABLE 5 Water uptake (%) of silk fibroin scaffolds. Particle size ofsodium Silk fibroin concentration (w/v %) chloride (μm) 4 6 8 101000~1180 98.2 ± 0.1 97.3 ± 0.1 95.9 ± 0.2 94.9 ± 1.0  850~1000 98.0 ±0.1 96.8 ± 0.1 95.2 ± 0.1 — 710~850 98.0 ± 0.1 96.7 ± 0.2 95.6 ± 0.4 —600~700 97.9 ± 0.1 96.6 ± 0.3 — — 500~600 97.9 ± 0.1 — — — Values areaverage ± standard derivation (N = 3).

Swelling ratio decreased gradually with a decrease in pore size.However, swelling ratio decreased significantly with an increase in silkfibroin concentration due to the decrease in porosity. The swellingratio of the scaffold prepared from 8 wt % silk fibroin was ˜8 timeslower than that of collagen scaffolds, due to the differences in thehydrophilicities of proteins (Ma L. et al., Biomaterials 2003,24:4833-4841). The value was similar to polylactic acid scaffolds(Maquet V. et al., Biomaterials 2004, 25:4185-4194). Water uptake of thescaffolds in distilled water was >93% during 24 hrs. The highwater-binding ability of the scaffolds can be attributed to the highlyporous structure of the protein network.

Mechanical Properties

The scaffolds exhibited a ductile and sponge-like behavior withdifferent stiffness depending on the concentration of silk fibroin usedin the process. An elastic region was observed at initial strainfollowed by a peak stress. Table 6 shows the mechanical properties ofthe silk fibroin scaffolds. The compressive strength and modulus of thescaffolds increased with an increase in silk fibroin concentration.

TABLE 6 Mechanical properties of silk fibroin scaffolds Particle Silkfibroin concentration (w/v %) size of 4 6 8 10 sodium CompressiveCompressive Compressive Compressive Compressive Compressive CompressiveCompressive chloride stress modulus stress modulus stress modulus stressmodulus (μm) (kPa) (kPa) (kPa) (kPa) (kPa) (kPa) (kPa) (kPa) 1000~118011 ± 3 70 ± 5 49 ± 4 560 ± 50 100 ± 10 1300 ± 40  320 ± 10 3330 ± 500 850~1000 11 ± 1 80 ± 5 54 ± 8 620 ± 40 125 ± 10 1530 ± 190 — 710~850 12± 1 100 ± 5  58 ± 3 670 ± 30 140 ± 15 1940 ± 240 — 600~700 13 ± 3 115 ±10 60 ± 5 770 ± 50 — — 500~600 13 ± 3 130 ± 10 — — — Values are average± standard derivation (N = 4).

The improvement in mechanical properties was attributed to the increasein polymer concentration accompanied with the increase in the wallthickness of the pores. At the same silk fibroin concentration,scaffolds prepared with smaller particle sizes of NaCl showed highercompressive strength and modulus due to the decreased pore size. It isconsidered that the increased pore wall sites induced by the decreasedpore size provided more paths to distribute the applied stress. Theincreased pore sites may functioned as a barrier, such as crackdisipation, to reduce crack propagation. In addition, it has beenreported that a more uniform pore distribution improved the mechanicalproperties of polymer matrices. Therefore, stress applied to porousmaterials is concentrated at the pore interface, and if the poredistribution is not uniform, polymer matrices typically deform at alower stress (Harris L D. Et al., J Biomed Mater Res 1998, 42:396-402).For example, in our recent studies (Nazarov R. et al.,Biomacromolecules, in press). three-dimensional silk fibroin scaffoldswere developed using a salt leaching method with HFIP. While thesescaffolds had smaller pore size and utilized a higher concentration ofsilk fibroin in processing, the compressive strengths (30 to 250 KPa) ofthe HFIP-derived silk scaffolds (17 wt % silk in HFIP) prepared by saltleaching were similar to those found for the aqueous-derived (8˜10 wt %silk in water) silk scaffolds in the present study. However, thecompressive modulus of aqueous-derived silk scaffolds was 3˜4 timeshigher (100 to 790 kPa) for the HFIP-derived silk scaffolds.

Enzymatic Degradation

FIG. 4 a shows the mass of the scaffolds over time prepared from 4 to 8wt % silk fibroin with NaCl of 850-1000 μm diameter particle sizesduring a degradation period of 21 days. The scaffolds in phosphatebuffer without protease showed no degradation within 21 days. Thescaffolds prepared with 4 wt % fibroin rapidly degraded and the massremaining was only 2% after 10 days. The scaffolds prepared from 6 and 8wt % fibroin gradually degraded with time and the mass was reduced to 30and 20%, respectively, after 21 days. FIG. 4 b shows the mass of thescaffolds remaining when prepared from 6 wt % silk fibroin with variousparticle sizes of NaCl. The degradation patterns suggest that pore sizedid not correlate with degradation rate, on the nature of the initialconcentration of fibroin.

Conclusions

Porous silk fibroin scaffolds were prepared directly from silk fibroinaqueous solutions by a salt leaching method, in the complete absence ofany organic solvents or chemical crosslinking. The formation of thescaffolds included a structural transition from random coil to β-sheet.This transition provides a mechanistic basis for the transition, as thesalt may promote water loss from the hydrophobic domains leading toenhanced chain-chain interactions and thus β-sheet formation. Functionaland morphological properties of the scaffolds were controlled by theconcentration of the silk fibroin solution used in the process and theparticle size of NaCl.

Example III Preparation of Silk Hydrogels

Control of silk fibroin concentration in aqueous solutions via osmoticstress was studied to assess relationships to gel formation andstructural, morphological and functional (mechanical) changes associatedwith this process. Environmental factors potentially important in the invivo processing of aqueous silk fibroin were also studied to determinetheir contributions to this process. Gelation of silk fibroin aqueoussolutions was affected by temperature, Ca²⁺, pH and polyethylene oxide(PEO). Gelation time decreased with increase in protein concentration,decrease in pH, increase in temperature, addition of Ca²⁺ and with theaddition of PEO. No change of gelation time was observed with theaddition of K⁺. Upon gelation, a random coil structure of the silkfibroin was transformed into β-sheet structure. Hydrogels with fibroinconcentrations >4 weight percent exhibited network and sponge-likestructures based on scanning electron microscopy. Pore sizes of thefreeze-dried hydrogels were smaller as the silk fibroin concentration orgelation temperature were increased. Freeze-dried hydrogels formed inthe presence of Ca²⁺ exhibited larger pores as the concentration of thision was increased. Mechanical compressive strength and modulus of thehydrogels increased with increase in protein concentration and gelationtemperature.

Methods Preparation of Silk Fibroin Aqueous Solution

Cocoons of Bombyx mori, kindly provided by M. Tsukada (Institute ofSericulture, Tsukuba, Japan) and M. Goldsmith (U. Rhode Island), wereboiled for 20 min in an aqueous solution of 0.02 M Na₂CO₃, and thenrinsed thoroughly with distilled water to extract the glue-like sericinproteins and wax. The extracted silk fibroin was then dissolved in 9.3 MLiBr solution at 60° C. for 4 hrs, yielding a 20 w/v % solution. Thissolution was dialyzed in distilled water using a Slide-a-Lyzer dialysiscassette (MWCO 3500, Pierce) for 2 days. The final concentration of silkfibroin aqueous solution was ca. 8 wt %, which was determined byweighing the remaining solid after drying. Silk fibroin film preparedfrom 8 wt % solutions was evaluated to verify the removal of Li⁺ ion byXPS; no residual Li⁺ ion was detected.

Preparation of Concentrated Silk Fibroin Solution by Osmotic Stress

Silk fibroin aqueous solution (8 wt %, 10 ml) was dialyzed against 10˜25wt % polyethylene glycol (PEG, 10,000 g/mol) solution at roomtemperature by using Slide-a-Lyzer dialysis cassettes (MWCO 3500). Thevolume ratio of PEG to silk fibroin solution was 100:1. By osmoticstress, water molecules in the silk fibroin solution moved into PEGsolution through the dialysis membrane (Parsegian, V. A., et al.,Methods in Enzymology, Packer, L., Ed.; Academic Press: 1986; Vol. 127,p 400). After the required time, concentrated silk fibroin solution wasslowly collected by syringe to avoid excessive shearing and theconcentration was determined. Silk fibroin aqueous solutions withconcentration less than 8 wt % were prepared by diluting 8 wt %solutions with distilled water. All solutions were stored at 7° C.before use.

Sol-Gel Transitions

A 0.5 ml of silk fibroin aqueous solution was placed in 2.5 mlflat-bottomed vials (diameter: 10 mm). The vials were sealed and kept atroom temperature, 37° C. and 60° C. Gelation time was determined whenthe sample had developed an opaque white color and did not fall from aninverted vial within 30 sec. To investigate the effect of ions and ionconcentration on the process, CaCl₂ or KCl solutions were added into thesilk fibroin aqueous solution to generate a final salt concentration of2.5 to 30 mM. The pH of the silk fibroin solution was adjusted with HClor NaOH solution. For the preparation of silk fibroin-poly(ethylene)oxide (PEO, 900,000 g/mol) solution, the required amount of PEO solution(5 wt %) was added to silk fibroin solution with mild stirring for 5minutes. The blend ratios of silk fibroin/PEO were 100/0, 95/5, 90/10,80/20 and 70/30 (w/w).

Wide Angle X-Ray Scattering (WAXS)

X-ray profiles were recorded for freeze-dried silk fibroin solutions andhydrogels using a Brucker D8 X-ray Diffractometer at 40 kV and 20 mA,with Ni-filtered Cu—Kα radiation.

Scanning Electron Microscopy (SEM)

Silk fibroin solutions and hydrogels were frozen at −80° C. and thenlyophilized. The samples were fractured in liquid nitrogen and examinedusing a LEO Gemini 982 Field Emission Gun SEM. To check for artifactualmorphological changes due to freeze-drying, an alternative preparationused Karnovsky's fixative at room temperature for 4 hrs. Hydrogels withand without fixative treatment showed little morphological change uponfreeze-drying. Pore size was obtained by using ImageJ software developedat the US National Institutes of Health.

Mechanical Properties

Compression tests of hydrogels were performed on an Instron 8511equipped with a 2.5 kN load cell at room temperature. A crosshead speedwas 10 mm/min. The cross-section of samples was 12 mm in diameter and 5mm in height. The compression test was achieved conventionally as anopen-sided method. The compression limit was 98% strain to protect theload cell. Five samples were evaluated for each composition.

Results Concentrated Silk Fibroin Solutions

Silk fibroin aqueous solution with an initial concentration of 8 wt %was dialyzed against 10˜25 wt % PEG solution at room temperature. Silkfibroin aqueous solution was concentrated over time by osmotic stressand concentrations of ca. 21 wt % were obtained after 9 hrs dialysisagainst 25 wt % PEG solution (FIG. 6). Longer dialysis times wererequired to generate higher concentrations of silk fibroin aqueoussolution, when lower concentrations of PEG solutions were used. Silkfibroin gels, 23˜33 wt %, were spontaneously generated in the dialysiscassettes during the concentration process. These gels were transparenteven after drying at room temperature and at 60° C.

Gelation of Silk Fibroin Aqueous Solution

The influence of temperature, Ca²⁺ and K⁺ concentrations, pH and PEOconcentration was investigated on the gelation of silk fibroin aqueoussolutions. FIG. 7 illustrates the gelation time of silk fibroin aqueoussolution (pH 6.5˜6.8) at various temperatures. The gelation time of silkfibroin aqueous solution decreased with increase in fibroin content andtemperature. Concurrently, a conformational change from random coil toβ-sheet structure was observed and the formation of β-sheet structure inthe hydrogels was confirmed by X-ray diffraction as described later.FIG. 8 shows the gelation time of silk fibroin aqueous solution withdifferent Ca²⁺ and K⁺ concentrations. The pHs of silk fibroin solutionswith Ca²⁺ and K⁺ ions were 5.6˜5.9 and 6.2˜6.4, respectively. Ca²⁺resulted in shorter gelation times, whereas there was no change ingelation time with the addition of K⁺ at any temperature. These resultswith regenerated silkworm fibroin differ from prior studies in which K⁺ions added to solutions of spider silk influenced aggregation andprecipitation of the protein, whereas there was no rheological changeafter addition of Ca²⁺ ions. FIG. 9 shows the gelation time of silkfibroin aqueous solution (4 wt %) at different pHs. Gelation timedecreased significantly with a decrease in pH. This behavior is similarto that observed for the silk from the spider, Araneus diadematus, whichgels at pH 5.5, but behaves as a viscous liquid at pH 7.4 (Vollrath, F.,et al., Proc. R. Soc. London B, 1998, 265, 817-820); FIG. 10 shows thegelation time of silk fibroin aqueous solution (4 wt %) with differentpolyethylene oxide (PEO) contents. By adding PEO solution, the pHdecreased slightly to the range 6.1˜6.4. The gelation time wassignificantly reduced with the addition of only 5% PEO, whereas therewas no difference in gelation time when the concentration was above 5%.

Structural Analysis of Hydrogels

Structural changes in the silk fibroin were determined by X-raydiffraction. FIG. 11 shows X-ray profiles of freeze-dried silk fibroinsolutions and hydrogels prepared from silk fibroin aqueous solutions.When silk fibroin solutions were frozen at low temperature, below theglass transition (−34˜−20° C.), the structure was not significantlychanged (Li, M., et al., J. Appl. Polym. Sci. 2001, 79, 2185-2191). Thefreeze-dried silk fibroin samples exhibited a broad peak at around 20°regardless of the silk fibroin concentration, indicating an amorphousstructure. Silk fibroin in aqueous solution at neutral pH exhibited arandom coil conformation. (Magoshi, J., et al., Polymeric MaterialsEncyclopedia; Salamone, J. C., Ed.; CRC Press: NewYork, 1996; Vol. 1, p.667; Magoshi, J., et al., Polymeric Materials Encyclopedia; Salamone, J.C., Ed.; CRC Press: NewYork, 1996; Vol. 1, p. 667). All hydrogelsprepared from silk fibroin solutions showed a distinct peak at 20.6° andtwo minor peaks at around 9° and 24°. These peaks were almost the sameas those of the β-sheet crystalline structure of silk fibroin. (Ayub, Z.H., et al., Biosci. Biotech. Biochem. 1993, 57, 1910-1912; Asakura, T.,et al., Macromolecules 1985, 18, 1841-1845). These peaks indicateβ-crystalline spacing distances of 9.7, 4.3 and 3.7 Å according to 9°,20.6° and 24°, respectively. From the results of X-ray diffraction, thegelation of silk fibroin solutions induced a conformational transitionfrom random coil to β-sheet as previously reported. (Ayub, Z. H., etal., Biosci. Biotech. Biochem. 1993, 57, 1910-1912; Hanawa, T., et al.,Chem. Pharm. Bull. 1995, 43, 284-288; Kang, G. D., et al., Marcromol.Rapid Commun. 2000, 21, 788-.791).

Morphology of Freeze-Dried Hydrogels

Morphological features of silk fibroin solutions and hydrogels wereobserved by SEM after freeze-drying at −80° C. Freeze-dried silk fibroinsolutions of 4˜12 wt % showed leaf-like morphologies. Freeze-dried silkfibroin solutions of 16 wt % and 20 wt % exhibited network andsponge-like structures with pore sizes of 5.0±4.2 μm and 4.7±4.0 μm,respectively. By SEM imagery it was determined that freeze-driedhydrogels prepared from 4 wt % silk fibroin solution showed leaf-likemorphologies and interconnected pores regardless of temperature and athigher fibroin concentrations than 4 wt % sponge-like structures wereobserved. The pore sizes of freeze-dried hydrogels (<1.1±0.8 μm) weresmaller than those observed for the freeze-dried silk fibroin solutionsamples. Pore sizes in the freeze-dried hydrogels decreased withincrease in silk fibroin concentration, and pore sizes decreased astemperature increased at the same silk fibroin concentration. The 4 wt %freeze-dried hydrogels with Ca²⁺ ions showed network and sponge-likestructures, whereas the 4 wt % freeze-dried hydrogels with K⁺ ions had aleaf-like morphology. In freeze-dried hydrogels with fibroinconcentrations >4 wt %, pore sizes of freeze-dried hydrogels with Ca²⁺were larger than those of freeze-dried hydrogels prepared from silkfibroin aqueous solutions without Ca²⁺ ions. Interestingly, pore sizewas larger in freeze-dried hydrogels with the same silk fibroinconcentration with an increase in Ca²⁺ concentrations. In contrast tofreeze-dried hydrogels with Ca²⁺, pore sizes of freeze-dried hydrogelswith K⁺ showed sizes similar to those of freeze-dried hydrogels preparedfrom silk fibroin aqueous solutions. These results imply that Ca²⁺ wasmore effective in inducing interactions among the silk fibroin chainsthan the K⁺. This result is also consistent with the earlier datawherein Ca²⁺ resulted in shorter gelation times than K⁺.

Mechanical Properties of Hydrogels

The compressive strength and modulus of hydrogels prepared from silkfibroin aqueous solutions increased with an increase in silk fibroinconcentration (FIGS. 12 a and 12 b). The improvement in mechanicalproperties was attributed to the increase in polymer concentrationaccompanied with the decrease in pore size. At the same silk fibroinconcentration, hydrogels prepared at higher temperature showed highercompressive strengths and moduli due to the decreased pore size.Hydrogels with 4˜8 wt % fibroin showed less than 55% strain, whilehydrogels with 12˜16 wt % fibroin revealed larger strains ranging from75% to 96% (FIG. 12 c). The effect of pore size was considered since thesmaller pore size distributes stress in the hydrogel more evenly toresist stress concentration. The smaller pore size and increased numberof pores also function as a barrier against crack propagation.

Discussion

Gelation occurs due to the formation of inter- and intramolecularinteractions among the protein chains, including hydrophobicinteractions and hydrogen bonds (Ayub, Z. H., et al., Biosci. Biotech.Biochem. 1993, 57, 1910-1912; Hanawa, T., et al., Chem. Pharm. Bull.1995, 43, 284-288; Kang, G. D., et al., Marcromol. Rapid Commun. 2000,21, 788-.791). With an increase in fibroin content and temperature,interactions among the fibroin chains increases. Silk fibroin moleculesare thereby able to interact more readily, leading to physicalcrosslinks.

The concentration of Ca²⁺ ion in the silkworm (Bombyx mori) increasesfrom 5 mM to 15 mM as silk progresses toward the spinneret, while K⁺ ionis present at 5˜8 mM.³ Several calcium salts are known to dissolve silkfibroin because of strong interactions with the fibroin (Ajisawa, A. J.Seric. Sci. Jpn. 1998, 67, 91-94; Ha, S. W., et al., Biomacromolecules2003, 4, 488-496). Rheological measurements of dilute solutions of silkfibroin from Bombyx mori revealed that the protein chains tend to formclusters by ionic interaction between COO⁻ ions of amino acid sidechains in the fibroin and divalent ions such as Ca²⁺ or Mg²⁺ (Ochi, A.,et al., Biomacromolecules 2002, 3, 1187-1196). Through theseinteractions, the pH of silk fibroin solutions with Ca²⁺ ions wassignificantly lower than that of silk fibroin solutions in the absenceof these ions, whereas the addition of monovalent ions such as K⁺ showedonly a slight decrease of pH. With lower pH, repulsion among silkfibroin molecules decreases and interactions among the chains is easier,resulting in stronger potential for the formation of β-sheet structurethrough hydrophobic interactions. A pH near the isoelectric point(pI=3.8-3.9) (Ayub, Z. H., et al., Biosci. Biotech. Biochem. 1993, 57,1910-1912; Kang, G. D., et al., Marcromol. Rapid Commun. 2000, 21,788-791) of silk fibroin accelerated the sol-gel transition of silkfibroin aqueous solutions in a fashion similar to other proteins thataggregate near their isoelectric points.

These outcomes may reflect subtle differences in how different silkproteins from different organisms utilize physiologically relevant ionsto facilitate sol-gel transitions. Divalent ions may induce aggregationof silk fibroin molecules by ionic interactions with negatively chargedamino acids present particularly near the chain ends of the heavy chainfibroin. The lack of response to different concentrations of Ca²⁺ maysuggest a broad window of response physiologically or perhaps a role forcombinations of ions to fully control this process in vivo or in vitro.Additional studies will be required to elucidate these relationships,particularly when considered in concert with observations on domainmapping of silks related to processing environments (Bini, E., et al.,J. Mol. Biol. 2004, 335, 27-40).

The movement of water from the silk fibroin molecules to the hydrophilicPEO facilitates inter- and intramolecular interactions among the proteinmolecules and the subsequent formation of the β-sheet structure. Thistransition is evident with silk based on our recent mechanisticunderstanding of the process (Jin, H. J., et al., Nature 2003, 424,1057-1061). These transitions can be induced by direct addition of PEOinto the fibroin aqueous solutions, or via separation from the aqueoussolutions across a dialysis membrane (with PEG). Thus, direct contactbetween the protein and the PEO is not required, only the facilitationof water transport from the protein to the PEO/PEG to drive the sol-geltransition.

Conclusions

From the primary sequence, silkworm silk fibroin heavy chain is composedof seven internal hydrophobic blocks and seven much smaller internalhydrophilic blocks, with two large hydrophilic blocks at the chain ends(Zhou, C. Z., et al., Nucleic Acids Res. 2000, 28, 2413-2419; Jin, H.J., et al., Nature 2003, 424, 1057-1061). The percentage of hydrophobicresidues in silk fibroin is 79% (Braun, F. N., et al., Int. J. Biol.Macromol. 2003, 32, 59-65) and the repetitive sequence in hydrophobicresidues consists of GAGAGS peptides that dominate the β-sheet structureforming crystalline regions in silk fibroin fibers and films (Mita, K.,et al., J. Mol. Evol. 1994, 38, 583-592). The formation of theseβ-sheets results in insolubility in water (Valluzzi, R., et al., J.Phys. Chem. B 1999, 103, 11382-11392). Hydrophobic regions of silkfibroin in aqueous solution assemble physically by hydrophobicinteractions and eventually organize into hydrogels (Jin, H. J., et al.,Nature 2003, 424, 1057-1061). Silk fibroin concentration, temperature,Ca²⁺, pH and PEO affected the gelation of the silk fibroin aqueoussolutions. With increase in fibroin content and temperature, physicalcrosslinking among silk fibroin molecules formed more easily. Ca²⁺ ionsaccelerated these interactions, presumably through the hydrophilicblocks at the chain ends. The decrease in pH and the addition of ahydrophilic polymer decreased repulsion between silk fibroin moleculesand promoted the desorption of water from the protein, resulting inshorter gelation times. Upon gelling, a conformational transition fromrandom coil to β-sheet structure was induced and promoted theinsolubility and stability of silk fibroin hydrogels in water. Silkfibroin hydrogels had network and sponge-like structures. Pore size wassmaller with increased silk fibroin concentration and gelationtemperature. Freeze-dried hydrogels showed larger pore sizes withincreases in Ca²⁺ concentrations than freeze-dried hydrogels preparedfrom silk fibroin aqueous solutions at the same fibroin content. Thecompressive strength and modulus of hydrogels prepared from silk fibroinaqueous solution without ions increased with increase in proteinconcentration and gelation temperature.

Hydrogels from natural polymers, such as collagen, hyaluronate, fibrin,alginate and chotosan, have found numerous applications in tissueengineering as well as in drug delivery. However, they generally offer alimited range of mechanical properties (Lee, K. Y., et al., Chem. Rev.2001, 101, 1869-1879). In contrast, silk fibroin provides an importantset of material options in the fields of controlled release,biomaterials and scaffolds for tissue engineering because of combinationwith impressive mechanical properties, biocompatibility,biodegradability and cell interaction (Altman, G. H., et al.,Biomaterials 2003, 24, 401-416; Cappello, J., et al., J. Control.Release 1998, 53, 105-117; Foo, C. W. P., et al., Adv. Drug Deliver.Rev. 2002, 54, 1131-1143; Dinerman, A. A., et al., J. Control. Release2002, 82, 277-287; Megeed, Z., et al., Adv. Drug Deliver. Rev. 2002, 54,1075-1091; Petrini, P., et al., J. Mater. Sci-Mater. M. 2001, 12,849-853; Altman, G. H., et al., Biomaterials 2002, 23, 4131-4141;Panilaitis, B., et al., Biomaterials 2003, 24, 3079-3085).

Example IV Bone Regeneration Using Three-Dimensional Aqueous-DerivedSilk Scaffolds

We have examined the bone regeneration of human bon marrow stem cells onthree-dimensional silk scaffolds from aqueous silk solutions of theinvention. To study the ability of the silk scaffolds to support thegrowth and differentiation of the bone marrow stem cells, we have usedthe silk scaffolds without any decoration.

Methods Materials

Bovine serum, Dulbecco's Modified Eagle Medium (DMEM), Minimal essentialmedium-α modification (αMEM), basic Fibroblast growth factor (bFGF),Penicillin-Streptomycin (Pen-Strep), Fungizone, non essential aminoacids, trypsin were from Gibco (Carlsbad, Calif.). Ascorbic acidphosphate, Histopaque-1077, dexamethasone, and β-glycerolphosphate werefrom Sigma (St. Lois, Mo.). All other substances were of analytical orpharmaceutical grade and obtained from Sigma. Silkworm cocoons werekindly supplied by M. Tsukada (Institute of Sericulture, Tsukuba, Japan)and Marion Goldsmith (University of Rhode Island, Cranston, R.I.).

Preparation of Scaffolds

Aqueous-derived silk scaffolds were prepared by adding 4 g of granularNaCl (particle size; 1000˜1180 μm) into 2 ml of 8 wt % silk fibroinsolution in disk-shaped Teflon containers. The container was covered andleft at room temperature. After 24 hrs, the container was immersed inwater and the NaCl was extracted for 2 days. HFIP-derived silk scaffoldswere prepared by adding 4 g of granular NaCl (particle size; 850˜100 μm)into 2 ml of 8 wt % silk fibroin in HFIP. The containers were covered toreduce of HFIP and to provide the sufficient time for more homogeneousdistribution of the solution. The solvent was evaporated at roomtemperature for 3 days. After the composite of silk/porogen was treatedin methanol for 30 min to induce β-sheet structure and insolubility inaqueous solution, the composite was immersed in water to remove NaCl for2 days. The porous silk scaffolds were air-dried.

Human Bone Marrow Stem Cell Isolation and Expansion

Total bone marrow (25 cm³, Clonetics, Santa Rosa, Calif.) was diluted in100 ml of isolation medium (5% FBS in RPMI 1640 medium). Cells wereseparated by density gradient centrifugation. Briefly, 20 ml aliquots ofbone marrow suspension were overlaid onto a poly-sucrose gradient (1,077g/cm³, Histopaque, Sigma, St. Louis, Mo.) and centrifuged at 800×g for30 min at room temperature. The cell layer was carefully removed, washedin 10 ml isolation media, pelleted and contaminating red blood cellswere lysed in 5 ml of Pure-Gene Lysis solution. Cells were pelleted andsuspended in expansion medium (DMEM, 10% FBS, 1 ng/ml bFGF, 100 U/mlpenicillin, 100 μg/m streptomycin, 0.25 μg/m fungizone, nonessentialamino acid) and seeded in 75 cm² flasks at a density of 5×104 cells/cm².The adherent cells were allowed to reach approximately 80% confluence(12-17 days for the first passage). Cells were trypsinized, replated andpassage 2 (P2) cells (80% confluence after 6-8 days), were used for theexperiments.

In Vitro Culture

For examination of cell growth and differentiation in vitro on silkscaffolds, BMSC (5×10⁵ cells/scaffold, passage 2) was seeded ontoprewetted (α-MEM, overnight) silk scaffolds. After 24 h, the medium wasremoved and cultures were maintained in individual wells of 6-wellplates. Osteogenic media were α-MEM supplemented with 10% FBS,nonessential amino acid, 50 μg/m ascorbic acid-2-phosphate, 10 nMdexamethasone, and 7 mM β-glycerolphosphate in the presence ofpenicillin and streptomycin and fungizone. Cultures were maintained at37° C. in a humidified incubator supplemented with 5% CO₂. Half of themedium was changed every 2-3 days.

Biochemical Analysis and Histology

Scaffolds were cultured for 2 and 4 weeks in osteogenic media andprocessed for biochemical analysis and histology. For DNA analysis, 3-4scaffolds per group and time point were disintegrated. DNA content(n=3-4) was measured using the PicoGreen assay (Molecular Probes,Eugene, Oreg.), according to the protocol of the manufacturer. Sampleswere measured flurometrically at an excitation wavelength of 480 nm andan emission wavelength of 528 nm. For total calcium content, samples(n=4) were extracted twice with 0.5 ml 5% trichloroacetic acid. Calciumcontent was determined by a colorimetric assay using o-cresolphthaleincomplexone (Sigma, St. Louis, Mo.). The calcium complex was measuredspectrophotometrically at 575 nm. Alkaline phosphatase activity wasmeasured using a biochemical assay from Sigma (St. Louis, Mo.), based onconversion of p-nitrophenyl phosphate to p-nitrophenol, which wasmeasured spectrophotometrically at 405 nm.

RNA Isolation, Real-Time-Reverse Transcription Polymerase Chain Reaction(Real Time RT-PCR)

Fresh scaffolds (n=3-4 per group) were transferred into 2 ml plastictubes and 1.0 ml Trizol was added. Scaffolds were disintegrated usingsteel balls and a Microbeater. Tubes were centrifuged at 12000 g for 10minutes and the supernatant was transferred to a new tube. Chloroform(200 μl) was added to the solution and incubated for 5 minutes at roomtemperature. Tubes were again centrifuged at 12000 g for 15 minutes andthe upper aqueous phase was transferred to a new tube. One volume of 70%ethanol (v/v) was added and applied to an RNeasy mini spin column(Quiagen, Hilden, Germany). The RNA was washed and eluted according tothe manufacturer's protocol. The RNA samples were reverse transcribedinto cDNA using oligo (dT)-selection according to the manufacturer'sprotocol (Superscript Preamplification System, Life Technologies,Gaithersburg, Md.). Collagen type I, Collagen type II, Alkalinephosphatase, bone sialoprotein and Osteopontin gene expression werequantified using the ABI Prism 7000 Real Time PCR system (AppliedBiosystems, Foster City, Calif.). PCR reaction conditions were 2 min at50° C., 10 min at 95° C., and then 50 cycles at 95° C. for 15 s, and 1min at 60° C. The expression data were normalized to the expression ofthe housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).The GAPDH probe was labelled at the 5′ end with fluorescent dye VIC andwith the quencher dye TAMRA at the 3′ end. Primer sequences for thehuman GAPDH gene were: Forward primer 5′-ATG GGG AAG GTG AAG GTC G-3′(SEG ID NO: 1), reverse primer 5′-TAA AAG CCC TGG TGA CC-3′ (SEQ ID NO:2), probe 5′-CGC CCA ATA CGA CCA AAT CCG TTG AC-3′(SEQ ID NO: 3).Primers and probes for alkaline phosphatase, bone sialoprotein (BSP),osteopontin and were purchased from Applied Biosciences (Assay on Demand#, Hs 00240993 ml (ALP), Hs 00173720 ml (BSP), Hs 00167093 ml(osteopontin)).

Western Blotting Analysis

For total protein extraction, cells were lysed in RIPA buffer [50 mMTris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.2% SDS, 5 mMNaF] containing protease inhibitors and phosphatase inhibitors. Proteincontent was measured by the Bradfod method. Proteins were resolved by3-8% SDS-PAGE and transferred to membranes. Blots were probed with theprimary antibody for 12 h at 4° C., washed, and incubated with theappropriate peroxidases labed secondary antibodies for 1 h at roomtemperature. Protein bands were revealed by ECL (Armersham-Pharmacia,)

Scanning Electron Microscopy (SEM) Analysis

The polymeric surfaces prior to and after cell attachment were examinedby scanning electron microscopy (SEM). Matrices were fixed usingKarnovsky's fixative for 24 h, and washed three times in CMPBS to removeresidual fixative. The samples were then dried using a graded series ofethanol (50˜100%) at 15 min intervals. After drying, the samples weresputter coated with gold and examined with a LEO Gemini 982 FieldEmission Gun SEM.

Histological Evaluation

After fixation with 4% phosphate-buffered formaldehyde for at least 24hours, specimens were embedded within paraffin and sectioned (4 μm).Using standard histochemical techniques, serial sections were stainedwith hematoxylin and eosin and alcian blue.

Results SEM Analysis

Characterization of the 3D-silk scaffolds was determined by assessmentof structurally by SEM for the analysis of pore size distribution andsurface topography. SEM analysis showed the water-silk scaffold had theinterconnected porous networks with an average pore size 920±50 μm. Poresurfaces had an appearance of a rough structure with nonhomogenousmicropores. However, HFIP-silk scaffold had the poor interconnectedporous networks with an average pore size 900±40 μm and showed thesmooth surface structure. BMSCs (passage 2) were seeded in water-silksponge and HFIP-silk sponge. More cells adhered to the water-silk spongethan to the HFIP-silk sponge. Water-silk sponge facilitated cellseeding. And BMSCs were homogeneously distributed throughout thewater-silk sponge. In contrast, the distribution of BMSCs on HFIP-silksponge was not homogenous. BMSCs growth was observed on water-silksponge. SEM confirmed extensive growth of BMSCs on water-silk sponge,followed by growth for up to 4 weeks.

Gross Examination

The cell-scaffold constructs were cultured in osteogenic media under a5% CO₂ atmosphere at 37° C. Constructs were cultured for up to 28 daysin 6 well plates. The BMSCs-water-silk constructs were became roundedafter culturing but BMSCs-HFIP-silk constructs were originally flat anddid not change after culturing. The tissue formed in the water-silkscaffold was whitish, hard to the touch and with surgical forceps. Butthe BMSCs seeded in the HFIP-silk scaffold did not form whitish tissue.No significant difference was noted 2- and 4-week specimens on grossexamination. And homogenous cellular distribution of BMSCs within thewater-silk scaffold was qualitatively demonstrated by the uniformity ofmatrix staining (indicative of MTT conversion by viable cells) on thesurface and throughout the center of the scaffold. But HFIP-silkscaffold displayed intense staining along the surface of construct andregions of weak staining within the interior of the construct.

Biochemical Analysis

The porosity of the 3D matrices was ca 92% in both water- and HFIP-silk.The compressive strength and modulus water-silk scaffolds were 100±10kPa and 1300±40 kPa. Those of HFIP-scaffolds were 50±5 kPa and 210±60kPa.

The total number of cells cultured on the scaffolds was quantified usinga DNA assay over the time course of the study. For water-silk scaffoldsseeded with cells suspended in medium, there was an increase from51,000±12,000 cells after initial seedings to 150,000±12,000 cells after28 days of culture. HFIP-silk scaffolds seeded with cells suspended inmedium did not show significant proliferation from the 8,000±3,400 cellsafter the initial cell seeding to 32,000±11,000 cells after 28 days ofculture.

Alkaline phosphatase (ALPase) activity, an indicator of theosteoprogenitor cell's commitment to the osteoblastic phenotype, wasmeasured on a per-scaffold. For water-silk scaffold, there was asignificant increase in ALPase activity after 28 days in culture(9.7±0.3 mmol/scaffold) compared to 1 day (0.4±0.01 mmol/scaffold).After 28 days of culture for HFIP-silk scaffold, 2.9±0.12 mmol perscaffold was detected.

The total calcium content of each sample was measured on a per-scaffold.Significant calcium deposition (10.5±0.65 μg/scaffold) was found after28 days of culture in osteogenic media for water-silk scaffold. After 28days of culture for HFIP-silk scaffold, there was 1.4±0.1 μg of Ca²⁺ perscaffold.

Expression of Osteogenic Differentiation Associated Genes

To characterize the bone-like tissue produced by BMSC, the expression ofseveral osteogenic differentiation and condrogenic differentiationmarker genes were quantified using real-time RT-PCR assays. The genesanalyzed included the osteogenic differentiation markers collagen type I(Col I), alkaline phosphatase (ALP), osteopontin (OP), bone sialoprotein(BSP), and the condrogenic differentiation marker collagen type II (ColII). The transcription level (normalized to GAPDH within the linearrange of amplification) differences between scaffold types weresignificant. Col I, ALP, and OP transcription levels increased inwater-silk scaffold when compared with HFIP-silk scaffold. After 28 daysof culture, gene expression of Col I, ALP and BSP was significantlyincreased by 190%, 1100% and 10500%, respectively, in water-silkscaffolds when compared with after 1 days of culture. However, OP andCol II expression was significantly decreased. BSP expression wasregulated similarly in water-silk scaffolds and HFIP-silk scaffolds. Thedifferences between scaffold types were not statistically significant.

Expression of Osteogenic Differentiation Associated Proteins

In 3-D water-silk scaffold culture condition, human bone marrow stemcells expressed the osteoblast markers. In comparison to HFIP-silkconstructs, the expression of Col I showed significant increase ofprotein levels under water-silk culture condition after 2 weeks. Howeverthe expression of Col I was decreased after 4 weeks culture under theboth conditions. After 28 days of culture, the expression of OP wasincreased in water-silk constructs. The protein showed two bands, ofwhich that at the highest molecular weight was assumed to be highlyglycosilated, sulfated or phosphorylated (Singh et al., J Biol Chem,1990, 65:18696-18701). The other protein of bone, BSP was expressed incells cultured both in water-silk scaffolds and in HFIP-silk scaffolds.However its expression was increased in HFIP-silk constructs after 28days of culture.

We also analyzed the expression of matrix metalloproteinase 13 (MMP13)and Col II. MMP13 was expressed in only water-silk constructs. And theCol II was downregulated in both culture conditions after 4 weeks.

Histological Examination

Histological examination of these specimens using hematoxylin and eosinstains revealed that the percentage of osteoblast-like cells in theircuboidal or columnar morphology increased with an increase in theculture period in water-silk constructs. After 14 days of culture,almost all pores were filled with connective tissue, fibroblasts andcuboidal osteoblast-like cells. After 28 days, the pores were filledwith extracellular matrix, osteoblast-like cells and few cells withfibroblast-like morphology. However, the histological sections ofHFIP-silk scaffolds demonstrated that there was a sparse distribution ofcells, with their majority forming a cell layer near the scaffold'ssurface. After 28 days of culture, the majority of cells withinHFIP-silk constructs displayed a flat fibroblastic morphology.

After culture in osteogenic media, extracellular matrices ofproteoglycan by alcian blue stains revealed that proteoglycans weredetected in water-silk constructs. No proteoglycn was histologicallydetected in HFIP-silk constructs.

Discussion

The silk protein-based matrix scaffolds are of current interest for bonetissue engineering. These scaffolds exhibit higher mechanical propertiesthan the other common biodegradable synthetic and natural polymers suchas PGA-PLA copolymers and collagen. HFIP have been used to prepareporous silk fibroin materials. Although HFIP-silk scaffolds are knownfor their unique mechanical properties, these natural polymers lackcell-recognition signals, which results in insufficient cell adhesion.To overcome this problem, a number of approaches have been developed,including: surface modification with arginine-glycine-aspartic acid(RGD), and hybrid with naturally derived biodegradable polymers.

Cell adhesion is known as an important cellular process because itdirectly influences cell growth and differentiation. In the presentexample, used the silk scaffolds without any modification. We observedsufficient cell adhesion in water-silk scaffolds. More cells adhered tothe water-silk scaffolds than to the HFIP-silk scaffolds. Variations insurface texture, or microtopography can affect the cellular response. Itwas found that a higher percentage of cells attached to the roughersurface. SEM analysis and histological analysis showed our water-silkscaffold had the rough structure with homogenous pores. However,HFIP-silk scaffold had the smooth surface structure.

With respect to the microstructures of the porous scaffolds, highporosity (>90%) and interconnected pore network are desirable. Inaddition, the preferred pore size is generally in the range of 50-500 μmto permit the ingrowth of cells and regeneration of tissue (Katoh K. etal., Biomaterials 2004, 25: 4255-4262; Thomson R, et al., In Principlesof Tissue Engineering; Lanza R, Langer R and Vacanti J, eds. AcademicPress: San Diego, pp. 251-262, 2000). The mitigation of nutrienttransport limitations, external to three-dimensional cell/polymerconstructs, influences the proliferation, differentiation, andexpression of osteoblastic markers of MSCs seeded on three-dimensionalscaffolds (Sikavitsas V I. Et al., J Biomed Mater Res 62: 136-148). Thestructural characterization showed that the pore size and the porosityof the water-silk sponge were controlled by the size of the NaClparticulates. We prepared the water-silk sponge with the regulated poresize (920±50 μm), which had more than 90% of the porosity. Furthermore,the pores were opened to the outside, interconnected and were uniformlydistributed throughout the sponges.

Porous silk scaffolds were seeded with human BMSCs, and BMSCs-silkconstructs were cultured for an extended period of 28 days in two modelsilk scaffolds (water-silk scaffolds and HFIP-silk scaffolds). The BMSCsseeded in water-silk scaffolds demonstrated accelerated proliferationduring the first 2 weeks of culture, and the strongest ALP activity andthe highest calcium deposition at the end of the culture period.

The onset of skeletogenesis, whether during fetal development or adultrepair, begins with the condensation of mesenchymal stem cells. Shortlyafter the condensation stage, cells in the central region of theaggregation begin to adopt a cartilaginous phenotype (Ferguson C. etal., Mech. Dev. 1999, 87: 57-66). The expression of Col II showed thisevent in our silk scaffolds. Our investigation of Col II showed thatdespite the gene expression of Col II in early stage, differentiatedBMSCs cultured in water-silk scaffolds maintained a differentiatedphenotype to the end of the culture period. In early stage, we observedCol II expression expression in HFIP-silk constructs, too. But the ColII gene expression and protein expression were significantly reduced.

Chondrocytes progress from a proliferative to a hypertrophic state. Themajority of hypertrophic chondrocytes are ultimately fated to undergoprogrammed cell death, which is accompanied by remodeling of theextracellular matrix (ECM) and the subsequent deposition of new bone(Gerber H. et al., Nat. Med. 1999, 5: 623-628). MMP13 regulatesremodeling of the hypertrophic cartilage matrix. The expression of MMP13 observed in water-silk constructs, but not in HFIP-silk constructs,indicating that the cells in water-silk scaffold were mature andhypertrophic chondrocytes.

The switch from a cartilage template to a bone during endochondral boneformation is not a mere switch of cell phenotypes. The cartilaginous ECMis then replaced by the bone ECM. Proteoglycan synthesis, the expressionof ALP, and the expression of type I collagen, but very little type IIcollagen, were detected in water-silk constructs. Although theexpression of ALP and type I collagen, the markers of osteoblasticdifferentiation, were significantly increased in water-silk constructs,the other protein of bone, BSP thought to comprise 8-12% of the totalnoncollagenous protein, was expressed similarly in water-silk scaffoldsand HFIP-silk scaffolds. Coexpression of type I and type II collagendemonstrated in a study (Nakagawa T. et al., Oral Diseases 2003, 9:255-263) showing that chondrocytes change their phenotype to produce thebone-like matrix and remain within the endochondral bone.

One of the osteoblast markers, OP, appears to be highly expressed at twostages of osteogenesis: at an early, proliferative stage and at a laterstage, subsequent to the initial formation of mineralized bone matrix(Yae, K L. et al., J. Bone Miner. Res. 1994, 9: 231-240). Early in theculture, the expression of OP was upregurated in water-silk constructs.These studies point to the usefulness of water-silk scaffolds in theinitial formation of bone tissue. Although type I collagen constitutesthe largest portion (90%) of the organic matrix in bone, it is notunique to this tissue. Proteoglycan, or at least their componentglycosaminoglaycan chains, have long been recognized as small butsignificant components of the mineralized bone matrix (Fisher L W. etal., J. Biol. Chem. 1982, 258: 6588-6594; Fedarko N S. Et al., J. Biol.Chem. 1990, 265: 12200-12209). After the culture in water-silkscaffolds, staining of sections with alcian blue stain clearlydemonstrated the presence of proteoglycan in the ECM. Proteoglycan canbe found in the cartilage. The origin and tissue specificity of theseporteoglycans have not been determined. In our study, the proteoglycanwas detected after 14 days of culture in water-silk constructs, but notin HFIP-silk constructs.

The references cited throughout the application are incorporated hereinby reference.

1-5. (canceled)
 6. A method for processing a silk film by contacting thefilm with water vapor in the absence of alcohol.
 7. The method of claim6, further comprising a step of forming the silk film from aconcentrated aqueous silk solution containing at least 10% silk fibroin.8. The method of claim 7, wherein the concentrated aqueous solutioncontains between about 10% and about 30% silk fibroin.
 9. The method ofclaim 6 or claim 7, further comprising a step of stretching the filmmono-axially or bi-axially.
 10. The method of claim 6 wherein the stepof contacting the silk film with water vapor comprises placing the silkfilm under vacuum in the presence of distilled water.
 11. The method ofclaim 7 wherein the step of forming the silk film comprises combiningthe aqueous silk solution with a biocompatible polymer.
 12. The methodof claim 11, wherein the biocompatible polymer is selected from thegroup consisting of polyethylene oxide (PEO), polyethylene glycol (PEG),collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate,clitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylacticacid, polyglycolic acid, polyhydroxyalkanoates, dextrans,polyanhydrides, and combinations thereof.
 13. The method of claim 11,wherein the film comprises from about 50 to about 99.99 part by volumeaqueous silk protein solution and from about 0.01 to about 50 part byvolume biocompatible polymer.
 14. The method of claim 6, wherein thecontacting is performed in the absence of any organic solvent.
 15. Themethod of claim 6, wherein the film is from about 60 to about 240 umthick.
 16. The method of claim 6, wherein the film comprises multiplelayers.
 17. A silk film treated with water vapor.
 18. The silk film ofclaim 17, prepared by the method of claim
 6. 19. The silk film of claim18, prepared from a concentrated aqueous solution containing at least10% silk fibroin.
 20. The method of claim 19, wherein the concentratedaqueous solution contains between about 10% and about 30% silk fibroin.21. The silk film of claim 17 or claim 19, which film has been stretchedmono-axially or bi-axially.
 22. The method of claim 6 wherein the stepof contacting the silk film with water vapor comprises placing the silkfilm under vacuum in the presence of distilled water.